| Objective:This study further explored the role of TIM3 in the pathogenesis of MDS patients in these two aspects: the effect of TIM3 expression on the prognosis of hematopoietic stem cells in MDS patients;the expression of TIM3 in T cell subsets.This study will provide a theoretical basis for the early identification of malignant MDS clones.Methods:The subjects were newly diagnosed MDS patients and healthy control volunteers from January 2013 to July 2017 in Tianjin Medical University General Hospital.Part I Flow cytometry was used to detect the percentage of bone marrow stem cells in the bone marrow of TIM3+ stem cells in 41 cases of MDS initially treated patients and 24 healthy controls.The survival status of the patients was followed up,and the conversion rate was counted.Finally,the effect of TIM3 expression on the prognosis and long-term survival of hematopoietic stem cells from MDS patients was evaluated.Part II Flow cytometry was used to detect the expression of TIM3 in peripheral blood T-lymphocyte subsets(Th1,Th2,Th17,Treg and CTL cells)in 44 patients with newly diagnosed MDS and 20 healthy controls.We detected the number of T cell subsets(Th1,Th2,Th17,Treg,and CTL cells)in patients with relatively low risk and relatively high risk MDS.We also detected the effect of TIM3 on the secretion of the Treg cytokine TGF-β and CTL cytokines perforin and granzyme B in the relatively low-risk group and the relatively high-risk group.Finally,we analyzed the effect of TIM3 expression on the prognosis of TDS patients with different risk MDS.Results:Part I The expression level of TIM3 in patients with MDS TIM3+CD34+CD38-Lin-hematopoietic stem cells was positively correlated with the patients’ IPSS-R score(r=0.44,p < 0.01).The expression level of TIM3 on hematopoietic stem cells was related to the karyotype of MDS patients,and gradually increased with the karyotypic variation(F=8.18,P<0.01).At the same time,the conversion rate of MDS patients increased with the increase of the expression level of TIM3 in CD34+CD38-Lin-hematopoietic stem cells in MDS patients.There was a significant difference in the survival time between the high expression group,and the low expression group of TIM3 in MDS patients(32months vs 17.5months,Chi square=6.24,df=1,p=0.01),and the median survival time decreased with the increase of TIM3 expression level.Part II Study of Th1 cells found that the ratio of Th1 cells to CD3+CD8-T cell subsets in patients with relatively low-risk MDS was significantly increased compared with healthy controls(12.57±0.48% vs 10.96±0.52%,p=0.02),The proportion of Th1 cells in CD3+CD8-T cells in the relatively high-risk group was significantly reduced compared with the healthy control group(4.40±0.52% vs 10.96±0.52%,p<0.01).At the same time,TIM3 expression was increased in Th1 cells in relatively low-risk MDS patients and relatively high risk MDS patients(19.53±2.51% vs 13.10±1.94%,p=0.04;49.56±4.41% vs 13.10±1.94%,p<0.01);The study of Th2 cells found that the proportion of Th2 cells in CD3+CD8-T cell subsets in the relatively high-risk group was significantly higher than that in the healthy control group(8.65±0.83% vs 5.97±0.63%,p<0.01).And TIM3 is not expressed on Th2 cells;The study of Th17 cells found that the ratio of Th17 cells to CD3+CD8-T cell subsets was significantly increased in the relatively low-risk and relatively high-risk MDS patients compared with healthy controls(2.86±0.30% vs 2.02±0.23%,p=0.03;4.29±0.28% vs 2.02±0.23%,p<0.01).The expression level of TIM3 in Th17 cells in relatively low risk and relatively high risk MDS patients was significantly lower than that in healthy controls(21.85 ± 2.68 vs 14.69 ± 2.28%,p<0.01;47.11±3.00% vs 14.69 ± 2.28%,p<0.01);The study of CD4+CD25+CD127dim+Treg cells found that the relative high-risk group Treg cells accounted for the percentage of CD4+ cells was significantly higher compared with the healthy control group(8.86±0.34% vs 5.21±0.40%,p<0.01).At the same time,the expression of TIM3 in Treg cells in the relatively low-risk and relatively high-risk MDS patients was significantly higher(22.25±3.31% vs 9.30±1.42%,p<0.01;45.59±5.87% vs 9.30±1.42%,p<0.01).In addition,the study also found that the level of TGF-β secreted by Tim3+ Treg cells increased with the increased risk of MDS patients,and there was a statistically significant difference between the relatively low-risk group and the relatively high-risk group compared with the healthy control group(20.11±3.20% vs 49.29±5.16% vs 8.38±1.35%,p<0.05);The study of CD3+CD8+ CTL cells found that the ratio of CTL cells to peripheral blood mononuclear cell in the relatively low-risk and relatively high-risk MDS patients was significantly reduced compared with the healthy control group(19.05±1.37% vs 24.03±1.83%,p=0.02;13.36%±1.16 vs 24.03±1.83%,p<0.01).At the same time,the expression of TIM3 in CTL cells of patients was reduced than relatively low-risk group(15.73±1.68% vs 6.57±0.90%,p<0.01).The expression of TIM3 was significantly higher in relatively high-risk MDS patients with(20.95±1.81% vs 6.57±0.90%,p<0.01).In addition,perforin and granzymesB secreted by TIM3+CTL cells from patients with MDS were less than those from TIM3-CTL cells(11.64±1.60% vs 30.46±3.55%,p<0.01;10.72±1.73% vs 16.94±2.48%,p=0.02).Conclusions:(1)The expression of TIM3 on hematopoietic stem cells in patients with MDS increases with the increased risk,and the higher the IPSS-R score,the higher the expression of TIM3 in hematopoietic stem cells.At the same time,the expression level of TIM3 correlates with the karyotype of MDS patients and gradually increases with the karyotypic deterioration.(2)The conversion rate of MDS patients increases with the expression level of TIM3 on hematopoietic stem cells.The median survival time of MDS patients decreases with the increase of TIM3 expression on hematopoietic stem cells.The expression level of TIM3 on hematopoietic stem cells in patients with MDS is of great significance in judging the survival time.(3)The number of Th1 cells were decreased in MDS patients,and gradually decreased with the increase in the relative risk of MDS patients;The number of Th2 cells decreased in patients with MDS and gradually decreased with the increase in the relative risk of MDS patients;The number of Treg and Th17 cells was elevated in MDS patients and gradually increased with the relative risk of MDS patients.In addition,the TGF-β secreted by Treg cells gradually increased with the relative risk of MDS patients;the levels of perforin and granzymeB secreted by CTL cells decreased with the relative risk of MDS patients.These results suggest that Th1,Th2,Th17,Treg and CTL cells are involved in the immune surveillance system imbalance in patients with myelodysplastic syndrome.(4)TIM3 is highly expressed on Th1,Th17,Treg,and CTL cells and is not expressed on Th2 cells.TIM3 negatively regulates immune cells in patients with MDS.TIM3 is high expressed on Treg cells and promotes the secretion of its cytokine TGF-β,thereby it may inhibit the number of Th1 cells.It is high expressed on CTL cells and inhibits the secretion of cytokine perforin and granzyme.The high expression of TIM3 in T-cell subpopulations conferred immune tolerance on MDS patients,resulting in the immune escape of malignantly cloned MDS hematopoietic stem cells. |
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